Functional specificity of the two retinoic acid receptor RAR and RXR families in myogenesis.

In C2 myoblasts, retinoic acid (RA) is an efficient inducer of both growth arrest and differentiation. These RA effects are mediated through at least two classes of retinoic acid receptors (RARs and RXRs), which belong to the nuclear receptor superfamily. To determine the role played by each RAR or RXR family in this model system, we have analysed the effects of RA in C2 myoblasts expressing a dominant negative RAR (dnRAR) or a dominant negative RXR (dnRXR). The stable expression of dnRAR or dnRXR in C2 cells delays the RA-induced growth arrest and differentiation, an effect which is more pronounced in C2-dnRXR myoblasts. Furthermore, the RA-inducible expression of MyoD gene is lost in C2-dnRXR but not in C2-dnRAR cells, indicating that each family of retinoid receptors RAR and RXR may regulate distinct subsets of RA-responsive genes. Finally, using C2 cell lines with different retinoid responsiveness, we provided evidence for a link between the RXR and MyoD families in the process of myogenic differentiation. These results illustrate a critical role for RA-receptors in RA-control of C2 myogenesis and provide tools for studying the function of RA and its receptors during vertebrate development.

RXR alpha is essential for mediating the all-trans retinoic acid-induced growth arrest of C2 myogenic cells.

In C2 muscle cells, retinoic acid (RA) induces growth arrest associated with terminal differentiation. These RA actions are presumed to be mediated through nuclear receptors (RARs and RXRs) that belong to the superfamily of ligand-dependent transcription factors. In this study, we have characterized a myogenic C2 subclone, that unlike parental cells, is resistant to growth inhibition and differentiation by RA. Examination of these RA-sensitive and resistant C2 cells for the expression of retinoid acid receptors revealed a lack of RXR alpha expression at the myoblast stage in resistant C2 cells. To determine the functions of RXR alpha, we introduced an RXR alpha expression vector into RA-resistant C2 cells by transient or stable transfections. Our results show that RXR alpha restores the response to RA in this subclone with respect to AP1 inhibition and growth arrest. These observations indicate that RXR alpha plays a crucial role in mediating RA induced growth arrest of C2 myogenic cells.

Retinoic acid receptors and muscle b-HLH proteins: partners in retinoid-induced myogenesis.

The results reported here indicate that retinoic acid (RA) induces growth arrest and differentiation only in MyoD-expressing muscle cells. Transient transfection assays reveal a functional interaction between MyoD, a key myogenic regulator and RA-receptors, principal mediators of RA actions. Interestingly, we demonstrate that RXR-MyoD-containing complexes are recruited at specific MyoD DNA-binding sites in muscle cells. Furthermore, we also demonstrate that RA-receptors and the muscle basic helix-loop-helix (b-HLH) proteins interact physically. Mutational analysis suggests that this interaction occurs via the basic region of muscle b-HLH proteins and the DNA-binding domain of RA-receptors and is important for functional interactions between these two families of transcription factors. In conclusion, these results highlight novel interactions between two distinct groups of regulatory proteins that influence cell growth and differentiation.

Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently.

MyoD, a member of the family of helix-loop-helix myogenic factors that plays a crucial role in skeletal muscle differentiation, is a nuclear phosphoprotein. Using microinjection of purified MyoD protein into rat fibroblasts, we show that the nuclear import of MyoD is a rapid and active process, being ATP and temperature dependent. Two nuclear localization signals (NLSs), one present in the basic region and the other in the helix 1 domain of MyoD protein, are demonstrated to be functional in promoting the active nuclear transport of MyoD. Synthetic peptides spanning these two NLSs and biochemically coupled to IgGs can promote the nuclear import of microinjected IgG conjugates in muscle and nonmuscle cells. Deletion analysis reveals that each sequence can function independently within the MyoD protein since concomittant deletion of both sequences is required to alter the nuclear import of this myogenic factor. In addition, the complete cytoplasmic retention of a beta-galactosidase-MyoD fusion mutant protein, double deleted at these two NLSs, argues against the existence of another functional NLS motif in MyoD.